Stability of flint through o-linked glycosylation

ABSTRACT

The invention relates to O-linked glycosylated FLINT polypeptides that comprise O-linked oligosaccharides at amino acid position 174 and/or 216 of SEQ ID NO: 1 (i.e. mature FLINT), compositions thereof that may comprise divalent metal cation, clinical and therapeutic uses thereof, and pharmaceutical formulations comprising said polypeptides.

[0001] This application claims priority of Provisional Application Ser.No. 60/169,412, filed Dec. 7, 1999.

[0002] A number of tumor necrosis factor receptor proteins (“TNFRproteins”) have been isolated in recent years, having many potentbiological effects. Aberrant activity of these proteins has beenimplicated in a number of disease states.

[0003] One such TNFR homologue, referred to herein as “Fas LigandInhibitory Protein,” or “FLINT”, binds Fas Ligand (FasL), therebypreventing the interaction of FasL with Fas (See WO 99/50413, WO00/58466, and WO 00/37094, the entire teachings of which areincorporated herein by reference).

[0004] Increased activation of the Fas-FasL signal transduction pathwayis implicated in a number of pathological conditions, including runawayapoptosis (Kondo et al., Nature Medicine 3(4):409-413 (1997); Galle etal., J. Exp. Med. 182:1223-1230 (1995)), and inflammatory diseaseresulting from neutrophil activation (Miwa et al.,. Nature Medicine4:1287 (1998)).

[0005] “Runaway apoptosis” is a level of apoptosis greater than normal,including apoptosis occurring at an inappropriate time. Pathologicalconditions caused by runaway apoptosis include, for example, organfailure in the liver, kidneys and pancreas. Inflammatory diseasesassociated with excessive neutrophil activation include sepsis, ARDS,SIRS and MODS.

[0006] Compounds such as FLINT and analogs thereof, which inhibit thebinding of Fas to FasL, and LIGHT to LTβR and/or TR2/HVEM receptors, canbe used to treat or prevent diseases or conditions that may beassociated with apoptosis and/or inflammation.

[0007] The therapeutic utility of FLINT could be enhanced bymodifications that improve pharmacological properties (e.g., enhancedpotency, stability, longer in vivo half-lives, and/or greater affinityfor FasL), pharmaceutical properties (e.g., decreased aggregation andsurface adsorption, increased solubility and ease of formulation) and/orchemical properties such as susceptibility to proteolysis. It is knownthat glycosylation can modulate structure and stability of a protein,influence the activity of signaling molecules, impact molecularrecognition, and affect the activity of enzymes (See e.g. P. Van denSteen et al. Critical Reviews in Biochemistry and Molecular Biology,33(3), 151-208; P. Rudd, Trends in Glycoscience and Glycotechnology, 11,1-21, 1999).

[0008] While FLINT is known to be potentially glycosylated (See e.g. WO99/50413, WO 99/14330), there is no current understanding as to specificglycosylation patterns nor the impact of specific glycosylationpatterns, on the stability of the molecule, or analogs thereof. Thetherapeutic utility of FLINT is likely to be impacted by theglycosylation pattern. For example, the potency and/or stability of themolecule could be directly impacted by glycosylation, as is known forother proteins. There is therefore a need to understand and to optimizethe glycosylation of FLINT and analogs thereof so as to achieve abetter, more cost-effective therapeutic protein. The invention disclosedherein relates to a solution to this problem. Specifically, shown hereinis that FLINT and analogs produced in mammalian cells have multipleglycosylation species, and that species having O-linked glycosylationare more stable to degradation.

[0009] The present invention addresses the need for a FLINT molecule,including analogs thereof, with enhanced stability. Specifically, theinvention relates to O-linked glycosylated FLINT polypeptides, N-linkedglycosylated FLINT polypeptides, and N/O-linked FLINT polypeptides, andmethods for producing and using same. The compositions of the presentinvention relate further to O-linked glycosylated analogs of FLINT,N/O-linked species, and N-linked glycosylated species of FLINT analogs,wherein one or more complex carbohydrate structures are linked to athreonine, serine, or asparagine residue. The FLINT polypeptides of theinvention provide enhanced physical, thermal, and conformationalstability to FLINT molecules. The FLINT proteins of the presentinvention are useful as pharmaceuticals and for treating a variety ofdiseases and conditions in mammals including humans.

[0010] In one embodiment, an oligosaccharide is O-linked to T216 of SEQID NO:1 (coincident site T245 of SEQ ID NO:3) and/or T174 of SEQ ID NO:1(coincident site T203 of SEQ ID NO:3).

[0011] In one embodiment, the invention relates to FLINT polypeptidescomprising O-linked oligosaccharides.

[0012] In another embodiment, the invention relates to FLINTpolypeptides comprising O-linked oligosaccharides complexed with adivalent metal cation.

[0013] In another embodiment, the present invention relates to FLINTpolypeptides comprising O-linked oligosaccharides wherein saidoligosaccharides are covalently attached at T216 of SEQ ID NO:1 (T245 ofSEQ ID NO:3) and/or T174 of SEQ ID NO:1.

[0014] In another embodiment the present invention relates to a FLINTanalog comprising N-linked oligosaccharides.

[0015] In another embodiment, the invention relates to a FLINT analogpolypeptide comprising O-linked and N-linked oligosaccharides whereinsaid O-linked oligosaccharides are located at T216 of SEQ ID NO:1 (T245of SEQ ID NO:3) and said N-linked oligosaccharides are located at N144of SEQ ID NO:1.

[0016] In another embodiment, the invention relates to a method forproducing a FLINT analog polypeptide comprising O-linkedoligosaccharides at T216 of SEQ ID NO:1 said method comprisingexpression of a vector encoding a protease resistant FLINT analog in asuitable mammalian cell line, for example, AV12, 293 EBNA, or CHO.

[0017] In another embodiment, the invention relates to a compositionsubstantially enriched in a FLINT analog polypeptide having O-linkedoligosaccharide covalently linked to T216 of SEQ ID NO:1.

[0018] In another embodiment the invention relates to therapeutic andclinical uses of a FLINT analog polypeptide substantially comprisingO-linked oligosaccharides at T216 to prevent or treat a disease orcondition in a mammal in need of such prevention or treatment.

[0019] In another embodiment the invention relates to therapeutic andclinical uses of a FLINT analog polypeptide of the present invention toprevent or treat diseases including acute lung injury (ALI), acuterespiratory distress syndrome (ARDS), ulcerative colitis, and tofacilitate organ preservation for transplantation, to inhibit Tlymphocyte activation, to prevent or treat chronic obstructive pulmonarydisease (COPD), and to prevent or treat pulmonary fibrosis (PF).

[0020] In another embodiment the present invention relates to apharmaceutical composition substantially comprising an T216 and/orT174/T216 O-linked glycosylated FLINT analog polypeptide of theinvention.

[0021] In another embodiment, the present invention relates to a methodto produce a FLINT analog resistant to proteolysis between position 218and 219 of SEQ ID NO:1, alternatively between position 247 and 248 ofSEQ ID NO:3, comprising the step of increasing O-linked glycosylation atposition T216 of SEQ ID NO:1 (alternatively, T245 of SEQ ID NO:3).

[0022] In another embodiment, the invention relates to a proteaseresistant FLINT analog produced by the method of enhancing O-linkedglycosylation at position T216 of SEQ ID NO:1.

[0023] FLINT polypeptide undergoes proteolysis in vivo to produce atleast two major peptide fragments. One of the fragments consists ofresidues 1 through 218 of SEQ ID NO:1 (alternatively residues 1 through247 of SEQ ID NO:3), termed herein “FLINT metabolite;” the otherconsists of residues 219 through 271 of SEQ ID NO:1 (alternativelyresidues 248 through 300 of SEQ ID NO:3). Cleavage at the 218 positionin vitro can be achieved when native FLINT (SEQ ID NO:3), or matureFLINT (SEQ ID NO:1), is treated with a trypsin-like enzyme, for example,thrombin, trypsin or other serine protease. Applicants have discoveredthat amino acid changes at position 218 render the molecule resistant toproteolysis at that position, as described in PCT application WO00/58466.

[0024] In another embodiment, the invention relates to a FLINT analogthat is resistant to proteolysis between positions 218 and 219 of SEQ IDNO:1, and/or between positions 247 and 248 of SEQ ID NO:3 in vivo and/orin vitro, said analog having enhanced O-linked glycosylation at T216.

[0025] In another embodiment, the invention relates to a FLINT analoghaving enhanced O-linked glycosylation at T216, said analog comprising apolypeptide that is at least about 95% identical; alternatively at least96% identical; alternatively at least 97% identical; alternatively atleast 98% identical; alternatively still, at least 99% identical withresidues 214 through 222 of SEQ ID NO:1 and/or residues 243 through 251of SEQ ID NO:3.

[0026] In another embodiment, the invention relates to a FLINT analogcomprising one, two, three, four, five, or more amino acidsubstitution(s), deletion(s), or addition(s) in the region comprisingamino acids 214-222 of SEQ ID NO:1 and/or amino acids 243-251 of SEQ IDNO:3.

[0027] The compositions of the present invention relate to O-linkedglycosylated species of FLINT, N/O-linked glycosylated species of FLINT,and N-linked glycosylated species of FLINT, wherein one or more complexcarbohydrate structures are linked to a threonine, serine, or asparagineresidue. In a preferred embodiment, an FLINT polypeptide comprisesoligosaccharide O-linked to T174 of SEQ ID NO:1.

[0028] In one embodiment, the invention relates to a FLINT fragment, forexample, the metabolite fragment comprising O-linked oligosaccharides.

[0029] In another embodiment, the invention relates to FLINT fragments,for example, the metabolite fragment, comprising O-linkedoligosaccharides said polypeptides complexed with a divalent metalcation.

[0030] In another embodiment, the present invention relates to a FLINTfragment, for example, the metabolite fragment, comprising O-linkedoligosaccharides wherein said oligosaccharides are covalently attachedat T174 of SEQ ID NO:1 (T203 of SEQ ID NO:3).

[0031] In another embodiment, the invention relates to a FLINT fragment,for example, the metabolite fragment, comprising O-linked and N-linkedoligosaccharides wherein said O-linked oligosaccharides are located atT174 of SEQ ID NO:1 (T203 of SEQ ID NO:3) and said N-linkedoligosaccharides are located at N144 of SEQ ID NO:1.

[0032] In another embodiment, the present invention relates to a FLINTfragment, for example, the metabolite fragment, comprising N-linkedoligosaccharide.

[0033] In another embodiment the invention relates to a FLINT fragment,for example, the metabolite fragment, comprising N-linkedoligosaccharide at position N144 of SEQ ID NO:1.

[0034] In another embodiment, the invention relates to a method forproducing a FLINT polypeptide or fragment thereof comprising O-linkedoligosaccharides at T174 and/or T216 of SEQ ID NO:1 said methodcomprising expression of a vector encoding FLINT in a suitable mammaliancell line, for example, AV12, 293 EBNA, or CHO.

[0035] In another embodiment, the invention relates to a method forproducing a FLINT polypeptide or fragment thereof comprising N-linkedand O-linked oligosaccharide at T174, and/or T216, and N-144 of SEQ IDNO:1.

[0036] In another embodiment, the invention relates to a compositionsubstantially enriched in a FLINT polypeptide or fragment, for examplethe metabolite fragment, having O-linked oligosaccharide covalentlylinked to T174 of SEQ ID NO:1.

[0037] In another embodiment, the present invention relates to acomposition substantially enriched in a FLINT polypeptide or fragment,for example the metabolite fragment, having N-linked oligosaccharide.

[0038] In another embodiment, the invention relates to a compositionsubstantially comprising a FLINT polypeptide or fragment, for examplethe metabolite fragment, having O-linked oligosaccharide covalentlylinked to T174 of SEQ ID NO:1, or T216 of SEQ ID NO:1.

[0039] In another embodiment the invention relates to therapeutic andclinical uses of a FLINT polypeptide or fragment, for example themetabolite fragment, comprising O-linked oligosaccharides, or N-linkedoligosaccharides, or N/O-linked oligosaccharides, to prevent or treat adisease or condition in a mammal in need of such prevention ortreatment.

[0040] In another embodiment the invention relates to therapeutic andclinical uses of a FLINT polypeptide or fragment, for example themetabolite fragment, of the present invention to prevent or treat adisease, for example, acute lung injury (ALI), acute respiratorydistress syndrome (ARDS), ulcerative colitis, and to facilitate organpreservation for transplantation, to inhibit T lymphocyte activation, toprevent or treat chronic obstructive pulmonary disease (COPD), and toprevent or treat pulmonary fibrosis (PF).

[0041] In another embodiment the present invention relates to apharmaceutical composition comprising an N-linked glycosylated FLINTpolypeptide or fragment, for example the metabolite fragment, of theinvention.

[0042] In another embodiment the present invention relates to apharmaceutical composition comprising an N/O-linked glycosylated FLINTpolypeptide or fragment, for example the metabolite fragment, of theinvention.

[0043] In one aspect, the FLINT polypeptide of the invention has anamino acid sequence of SEQ ID NO:1, modified by:

[0044] a) replacing tryptophan at position 53 with aspartic acid;

[0045] b) replacing threonine at position 88 with proline;

[0046] c) replacing alanine at position 107 with serine, aspartic acid,glutamic acid or threonine;

[0047] d) replacing isoleucine at position 110 with threonine orglutamic acid; or

[0048] e) replacing proline at position 104 with serine.

[0049] In another aspect, the FLINT polypeptide has an amino acidsequence of SEQ ID NO:1, modified by:

[0050] a) replacing alanine at position 2 or position 12 withasparagine;

[0051] b) replacing proline at position 25, position 38, position 126 orposition 171 with asparagine;

[0052] c) replacing arginine at position 35 with asparagine;

[0053] d) replacing serine at position 37 with asparagine and proline atposition 38 with any other naturally occurring amino acid;

[0054] e) replacing serine at position 166 with asparagine;

[0055] f) replacing leucine at position 172 with asparagine;

[0056] g) replacing aspartic acid at position 194 with asparagine;

[0057] h) replacing threonine at position 114 with asparagine andproline at position 115 with any naturally occurring amino acid; or

[0058] i) replacing arginine at position 218 with asparagine.

[0059] In yet another aspect, the FLINT polypeptide has an amino acidsequence of SEQ ID NO:1, modified by:

[0060] a) replacing asparagine at position 63 with tryptophan;

[0061] b) replacing glycine at position 67 with aspartic acid andreplacing alanine at position 94 or glycine at position 95 withtyrosine;

[0062] c) replacing arginine at position 69 with glutamic acid;

[0063] d) replacing arginine at position 82 with glutamic acid orthreonine;

[0064] e) replacing alanine at position 94 with tyrosine and replacingglycine at position 95 with aspartic acid;

[0065] f) replacing phenylalanine at position 96 with glutamine;

[0066] g) replacing alanine at position 101 with threonine; or

[0067] h) replacing glycine at position 95 with aspartic acid.

[0068] In yet another aspect, the FLINT polypeptide has an amino acidsequence of SEQ ID NO:1, modified by:

[0069] a) replacing arginine at position 10 with glutamine, asparagine,serine or threonine, provided that when the replacing amino acid isasparagine, then alanine at position 12 is optionally replaced withserine or threonine;

[0070] b) replacing glutamic acid at position 13 with glutamine,asparagine, serine or threonine, provided that when the replacing aminoacid is asparagine, then glycine at position 15 is optionally replacedwith serine or threonine;

[0071] c) replacing glutamic acid at position 16 with glutamine,asparagine, serine or threonine, provided that when the replacing aminoacid is asparagine, then leucine at position 18 is optionally replacedwith serine or threonine;

[0072] d) replacing arginine at position 17 with glutamine, asparagine,serine or threonine, provided that when the replacing amino acid isasparagine, then valine at position 19 is optionally replaced withserine or threonine;

[0073] e) replacing arginine at position 31 with glutamine, asparagine,serine or threonine, provided that when the replacing amino acid isasparagine, then cysteine at position 33 is optionally replaced withserine or threonine;

[0074] f) replacing arginine at position 34 with glutamine, asparagine,serine or threonine, provided that when the replacing amino acid isasparagine, then aspartic acid at position 36 is optionally replacedwith serine or threonine;

[0075] g) replacing arginine at position 35 with glutamine, asparagine,serine or threonine;

[0076] h) replacing aspartic acid at position 36 with glutamine,asparagine, serine or threonine, provided that when the replacing aminoacid is asparagine, then proline at position 38 is optionally replacedwith serine or threonine;

[0077] i) replacing arginine at position 143 with glutamine, asparagine,serine or threonine, provided that when the replacing amino acid isasparagine, then cysteine at position 145 is optionally replaced withserine or threonine; or

[0078] j) replacing aspartic acid at position 161 with glutamine,asparagine, serine or threonine, provided that when the replacing aminoacid is aspargine, then leucine at position 163 is optionally replacedwith serine or threonine.

[0079] In yet another embodiment, the FLINT of the present invention isa polypeptide having the amino acid sequence of SEQ ID NO:1 modified by:

[0080] a) replacing alanine at position 2, 12, 107, 179 or 209 withthreonine;

[0081] b) replacing threonine at position 4 or 162 with alanine;

[0082] c) replacing valine at position 1 or isoleucine at position 110with methionine;

[0083] d) replacing glutamic acid at position 13 with aspartic acid;

[0084] e) replacing arganine at position 17 with tryptophan;

[0085] f) replacing alanine at position 75 with proline;

[0086] g) replacing serine at positione 102 with leucine;

[0087] h) replacing glycine at position 169 with alanine;

[0088] i) replacing glutamic acid at position 183 with lysine;

[0089] j) replacing glutamine at position 225 with arginine;

[0090] k) replacing glycine at position 237 with glutamic acid; or

[0091] l) replacing valine at position 270 with glycine, said fragmentcomprising amino acids 49-165 of the polypeptide; and physiologicallyacceptable salts thereof.

[0092] In yet another aspect, the FLINT polypeptide has an amino acidsequence of SEQ ID NO:1, modified by:

[0093] a) replacing alanine at position 12 with asparagine andoptionally replacing glutamic acid at position 13 with glutamine;

[0094] b) replacing arginine at position 34 with asparagine andreplacing aspartic acid at position 36 with threonine;

[0095] c) replacing arginine at position 35 with asparagine andoptionally replacing serine at position 37 with threonine;

[0096] d) replacing serine at position 132 with asparagine andoptionally replacing serine at position 134 with threonine;

[0097] e) replacing aspartic acid at position 194 with asparagine andoptionally replacing serine at position 196 with threonine;

[0098] f) replacing arginine at position 35 and aspartic acid atposition 194 with asparagine;

[0099] g) replacing alanine at position 12 with asparagine, optionallyreplacing glutamic acid at position 13 with glutamine, replacingaspartic acid at position 194 with asparagine and optionally replacingserine at position 196 with threonine;

[0100] h) replacing arginine at position 34 with asparagine, replacingaspartic acid at position 36 with threonine, replacing aspartic acid atposition 194 with asparagine and optionally replacing serine at position196 with threonine;

[0101] i) replacing arginine at position 35 and aspartic acid atposition 194 with asparagine and replacing serine at position 37 and/orposition 196 with threonine; or

[0102] j) replacing arginine at position 218 with glutamine.

[0103] k) replacing glycine at position 26 with aspartic acid andreplacing serine at position 132 with asparagine;

[0104] l) replacing alanine at position 12 with asparagine, replacingserine at position 132 with asparagine, and replacing serine at position134 with threonine; or

[0105] m) replacing threonine at position 216 with proline and replacingarginine at position 218 with glutamine.

[0106] O-linked, and/or N-linked, and/or N/O-linked glycosylated speciesof FLINT can be produced in mammalian cells transfected with anexpression vector that encodes FLINT. Applicants have observed variationin the glycosylation pattern of FLINT molecules produced thereby, forexample, in the fraction of polypeptides that are O-linked glycosylatedat T174. The fraction of such species may vary depending upon cellculture conditions and on the particular cell type used as host. In apreferred embodiment, a FLINT analog is expressed in CHO cells, fromwhich about 40% of the FLINT polypeptide produced is glycosylated atT174.

DETAILED DESCRIPTION OF THE INVENTION

[0107] SEQ ID NO:1—Mature human FLINT, i.e. native FLINT minus theleader sequence.

[0108] SEQ ID NO:2—Nucleic acid/cDNA encoding mature human FLINT.

[0109] SEQ ID NO:3—Native human FLINT.

[0110] SEQ ID NO:4—Human FLINT leader sequence.

[0111] SEQ ID NO:5—Oligonucleotide primer A, CF107

[0112] SEQ ID NO:6—Oligonucleotide primer B, CF111

[0113] SEQ ID NO:7—Oligonucleotide primer C, CF112

[0114] SEQ ID NO:8—Oligonucleotide primer D, CF110

[0115] SEQ ID NO:9—Nucleic acid encoding human FLINT.

[0116]FIG. 1. Oligosaccharide structures at T174 of native FLINT.

[0117]FIG. 2. Reverse phase chromatographic analysis of native FLINTglycosylation variants from AV12 cell line showing both N-linkedglycosylation at Asn 144 and O-linked glycosylation at Thr174 (peak A)and N-linked only (peak B).

[0118]FIG. 3. Reverse phase HPLC chromatogram of FLINT-A (N/O-linkednative FLINT) and FLINT-B (N-linked native FLINT).

[0119]FIG. 4. Reverse phase HPLC chromatogram of FLINT metabolitepurified after thrombin cleavage of wt FLINT using a C4 Vydac column.

[0120]FIG. 5. RP-HPLC chromatogram of FLINT metabolite afterconcentration step.

[0121]FIG. 6. Thermal denaturation of FLINT metabolite monitored bydifferential scanning calorimetry. The sample containing 0.2 mg/mL FLINTmetabolite was dialyzed against PBS at pH 7.2.

[0122] The term “analog” or “FLINT analog” is used herein specificallyto mean a FLINT molecule or sequence variant thereof having one or moreamino acid sequence changes, e.g. substitution, addition, deletion,including variants that are resistant to proteolysis between positions218 and 219 of SEQ ID NO:1 and/or positions 247 and 248 of SEQ ID NO:3.

[0123] The term “native FLINT” refers to SEQ ID NO:3.

[0124] The term “mature FLINT” refers to SEQ ID NO:1.

[0125] The term “FLINT” is used herein generically to encompass nativeand mature FLINT, FLINT fragments, and analogs of FLINT having analtered amino acid sequence comprising one or more amino acidsubstitutions, deletions, or additions.

[0126] The term “FLINT metabolite” or “metabolite” refers to a fragmentof FLINT comprising residues 1 to 218 of SEQ ID NO:1.

[0127] The term “N-glycosyled polypeptide” refers to polypeptides havingone or more NXS/T motifs in which the nitrogen atom in the side chainamide of the asparagine is covalently bonded to a glycosyl group. “X”refers to any naturally occurring amino acid residue except proline. The“naturally occurring amino acids” are glycine, alanine, valine, leucine,isoleucine, proline, serine, threonine, cysteine, methionine, lysine,arganine, glutamic acid, asparatic acid, glutamine, asparagine,phenylalanine, histidine, tyrosine and tryptophan. N-Glycosylatedproteins are optionally O-glycosylated. The term “O-glycosyledpolypeptide” refers to polypeptides having one or more serines and/orthreonine in which the oxygen atom in the side chain is covalentlybonded to a glycosyl group. O-Glycosylated proteins are optionallyN-glycosylated.

[0128] The term “substantially pure” or “substantially enriched” or“substantially comprising” is used herein to mean greater than 50%,preferably 80%-85%; most preferable at least 85% pure, i.e. separatedfrom other proteins and/or other FLINT glycosylation species, and/orcompounds and impurities. For example, a pharmaceutical compositionsubstantially comprising FLINT having O-linked glycosylation would referto a composition comprising a preparation of FLINT in which greater than50%, preferably 80%-85%, more preferably at least 85% of said FLINTcomprised O-linked glycosylated species.

[0129] The nucleotide and amino acid abbreviations used herein are thoseaccepted in the art and by the United States Patent and TrademarkOffice, as set forth in 37 C.F.R. 1.822 (b)(2).

[0130] Descriptions herein relating to carbohydrate content of a FLINTpolypeptide at position T174 (O-linked), T216 (O-linked), and N144(N-linked) of SEQ ID NO:1 (mature FLINT) are intended also to relate toSEQ ID NO:3 (native FLINT having leader sequence), wherein the positionof carbohydrate content is at 203, 245, and 173, respectively.

[0131] Applicants have discovered that mutations in FLINT that renderthe molecule resistant to proteolysis between the arginine residue atposition 218 and the alanine residue at position 219 of SEQ ID NO:1,concomitantly increase the O-linked glycosylation at T216.

[0132] Therefore, the invention further contemplates amino acid changesin the region from about position 214 through position 222 of SEQ IDNO:1 or the comparable region of SEQ ID NO:3 that increase theglycosylation at T216.

[0133] In one embodiment, a single amino acid change is made within theregion 214-222; alternatively, at least two changes are made within thisregion; alternatively, at least three changes are made within thisregion; alternatively, at least four changes are made within thisregion.

[0134] In one embodiment, the invention relates to a method forproducing a FLINT analog that is resistant to proteolysis betweenpositions 218 and 219 of SEQ ID NO:1 (positions 247 to 248 of SEQ IDNO:3), said method comprising the step of enhancing or increasingO-linked glycosylation at position T216 and/or T174 of SEQ ID NO:1 (T245and T203 of SEQ ID NO:3 respectively). Said enhancement or increase ofO-linked glycosylation can be achieved by changing the cell line inwhich a recombinant FLINT is expressed. For example, the level ofO-linked glycosylation of native FLINT was found to decrease in theorder CHO >293 >AV12. Alternatively, enhancement can be achieved bychanging the conditions under which a recombinant cell line expressingFLINT is grown. Alternatively still, O-linked glycosylation at T216 canbe enhanced by changing the arginine residue at position 218 to aneutral or negatively charged residue. Moreover, changing arginine, apositively charged amino acid, to a neutral or negatively charged aminoacid, increases the O-linked glycosylation at T216. For example, R218Eis O-linked glycosylated at T216 to a greater extent than R218Q, which,in turn, is O-linked glycosylated at T216 to a greater extent thannative FLINT (data not shown).

[0135] Applicants have observed variation in the glycosylation patternof FLINT molecules produced in different cell lines, for example, in thefraction of native FLINT polypeptides that are O-linked glycosylated atT174 and in the composition of oligosaccharides at these positions (SeeFIG. 1). In a preferred embodiment, FLINT is expressed in CHO cells fromwhich about 50% of the FLINT polypeptide produced is N/O-linkedglycosylated at T174. Other cell lines such as AV12 and 293 producedless than 50% N/O-linked species.

[0136] O-linked, N-linked, and N/O-linked FLINT polypeptides can beseparated from each other and from other glycosylation species of FLINTby a variety of suitable purification techniques, for example, reversephase HPLC and polyacrylamide gel electrophoresis. Differentglycosylation species of FLINT can be resolved by a variety ofanalytical and preparative techniques. For example, differentglycosylation species of FLINT prepared from a mammalian cell line, forexample, transfected AV12 cells, can be separated by PAGE or RP-HPLCrevealing at least two peaks (See FIG. 2).

[0137] Preferred O-linked FLINT polypeptides are glycosylated at T174and/or T216. O-linked glycosylated FLINT of the invention may also beN-linked glycosylated at position N144. FLINT polypeptides that areexpressed in mammalian cells such as 293 EBNA, AV12, and CHO aresubstantially glycosylated at N144, and partially O-linked glycosylatedat T174 and T216. The fraction of FLINT molecules that are O-linkedglycosylated at T216 tends to be less than at T174.

[0138] The O-linked glycosylated FLINT polypeptides of the inventioncomprise a plurality of O-linked oligosaccharide structures (See FIG.1). Predominant oligosaccharide structures at T216 and T174 on FLINTinclude GalNAc, Galactose, and NeuAc.

[0139] Stability of O-linked FLINT Molecules

[0140] It is generally known that glycosylation can effect the stabilityof protein molecules. The invention described herein relates, in oneembodiment, to FLINT polypeptides having increased stability, saidpolypeptides having O-linked oligosaccharides at T174 and/or T216, andN-linked oligosaccharides at N144. Preferably, said FLINT is O-linkedglycosylated at T174 and/or T216.

[0141] In another embodiment, the invention provides a compoundsubstantially comprising T216 O-linked glycosylated FLINT, alternativelyan N-linked FLINT, wherein said glycosylation occurs at, for example,T174 and/or T216, complexed with a divalent metal cation. In anotherembodiment, the invention provides a compound comprising an N-linkedFLINT protein, for example, N144 complexed with a divalent metal cation.In another embodiment, the invention provides a compound comprising anN/O-linked FLINT protein, for example, N144/T174, N144/T174/T216, orN144/T216, complexed with a divalent metal cation.

[0142] In another aspect, pharmaceutical compositions comprisingO-linked, or N-linked, or N/O-linked glycosylated FLINT and divalentcation(s) are provided. These compositions may be used in depotformulations for therapeutic application.

[0143] According to this embodiment, FLINT compositions compriseO-linked, or N-linked, or N/O-linked glycosylated FLINT polypeptidescomplexed with one or more divalent metal cations. Suitable divalentmetal cation include, for example, Zn⁺², Mn⁺², Fe⁺², Co⁺², Cd⁺², Ca⁺²,Ni⁺² and the like. Such compositions may comprise a single species ofmetal ion or a combination of two or more species of divalent metalcations. Preferred compounds comprise a single species of metal cation,most preferably Zn⁺². Preferably, the divalent metal cation is inexcess; however, the molar ratio of at least one molecule of a divalentmetal cation for each ten molecules of FLINT is operable. Preferably,the compounds comprise from 1 to 100 divalent metal cations per moleculeof FLINT. The compounds may be amorphous or crystalline solids.

[0144] Appropriate forms of metal cations are any form of a divalentmetal cation that is available to form a complex with a molecule of aFLINT protein of the present invention. The metal cation may be added insolid form or as a solution. Several different cationic salts can beused in the present invention. Representative examples of metal saltsinclude the acetate, bromide, chloride, fluoride, iodide and sulfatesalt forms. The skilled artisan will recognize that there are many othermetal salts which also might be used in the production of the compoundsof the present invention. Preferably, zinc acetate or zinc chloride isused to create the zinc-FLINT protein compounds of the presentinvention. Most preferably, the divalent metal cationic salt is zincchloride.

[0145] The present invention relates further to the use of the FLINTpolypeptides of the invention to inhibit apoptosis and/or T cellactivation. T cell activation can be chronically suppressed whenadvantageous, for example, following organ transplantation to preventrejection, in the treatment of autoimmune diseases, and in treatingsystemic inflammatory responses.

[0146] FLINT polypeptides of the invention can be produced byrecombinant techniques or by direct chemical synthesis, well known tothe skilled artisan. See. e.g. K. Struhl, “Reverse biochemistry: Methodsand applications for synthesizing yeast proteins in vitro,” Meth.Enzymol. 194, 520-535. In a preferred recombinant method, site-directedmutagenesis is used to introduce defined changes into the region 214-222of SEQ ID NO:1 or the comparable region of SEQ ID NO:3.

[0147] FLINT polypeptides also include modified derivatives thereof inwhich one or more polyethylene glycol groups (hereinafter “PEG” groups)are bonded to the N-terminus or to amine groups or thiol groups in theamino acid side chain(s). Suitable PEG groups generally have a molecularweight between about 5000 and 20,000 atomic mass units. Procedures forpreparing PEGylated polypeptides are disclosed in Mumtaz and Bachhawat,Indian Journal of Biochemistry and Biophysics 28:346 (1991) andFranciset al., International Journal of Hematology 68:1 (1998), theentire teachings of which are incorporated herein by reference.

[0148] Another embodiment of the invention relates to a fusion proteincomprising an O-linked FLINT polypeptide, or an N-linked FLINTpolypeptide, or an N/O-linked FLINT polypeptide. Preferred embodimentsinclude T174, T216, T174/T216, N144, T174/N144, T216/N144,T174/T216/N144. “Fusion protein” denotes a hybrid protein molecule notfound in nature comprising a translational fusion or enzymatic fusion inwhich two or more different proteins or fragments thereof are covalentlylinked on a single polypeptide chain. Human serum albumin and theC-terminal domain of thrombopoietin are examples of proteins which couldbe fused with a FLINT polypeptide of the invention. Procedures forpreparing fusion proteins are disclosed in EP394,827, Tranecker et al.,Nature 331:84 (1988) and Fares, et al., Proc. Natl. Acad. Sci. USA89:4304 (1192), the entire teachings of which are incorporated herein byreference.

[0149] A fusion protein of the present invention comprises two proteinsegments fused together by means of a peptide bond. The first proteinsegment consists of at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40,50, 75, 100, 125, 150, 175, 200, 225, 250, or 271 contiguous amino acidresidues of an FLINT polypeptide of the present invention (i.e.derivative of SEQ ID NO:1 or SEQ ID NO:3) having N-linked, or O-linked,or N/O-linked glycosylation; preferably N-linked at N144; O-linked at,alternatively, T174, T216, or T174/T216; and N/O-linked at T174/N144,T216/N144, or T174/T216/N144. The first protein can alternatively be afull length protein of the present invention and/or an N-terminal orC-terminal fragment thereof.

[0150] The second protein of a FLINT fusion protein of the invention canbe a full length protein or a protein fragment. Proteins commonly usedin fusion proteins include B-galactosidase, B-glucuronidase, greenfluorescent protein (GFP), thrombopoietin (TPO),glutathione-S-transferase (GST), luciferase, horseradish peroxidase, andchloramphenicol acetyltransferase (CAT). Epitope tags can be used infusion protein constructions including histidine (His) tags, FLAG tags,influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxintags. Other fusion constructions can include maltose binding protein,S-tag, Lex A DNA binding domain, GAL4 DNA binding domain fusions, andherpes simplex virus BP16 protein fusions.

[0151] The skilled artisan understands that nucleic acids encoding aFLINT (native or analog) of the present invention can be preparedsynthetically. For analogs, this can be achieved by mutating a nucleicacid template that encodes native FLINT, e.g. introducing appropriatepoint mutations into a cDNA encoding FLINT using any number of suitablemutagenic techniques known to the skilled artisan. Alternatively, saidnucleic acids can be prepared synthetically de novo based on knowledgeof the genetic code and the particular analog of SEQ ID NO:1 or SEQ IDNO:3 desired. Codon preference may be taken into account when designinga suitable nucleic acid.

[0152] A FLINT cDNA can be synthesized by RT-PCR using conventionaltechniques. For example, PolyA RNA is prepared from a tissue known toexpress the FLINT gene (e.g. human lung), using standard methods. Firststrand FLINT cDNA synthesis is achieved in a reverse transcriptasereaction using a FLINT sequence derived “downstream” primer. Acommercially available kit such as GENEAMP by Perkin Elmer may beemployed. In a subsequent PCR, FLINT specific forward and reverseprimers are used to amplify the cDNA. The amplified sample may beanalyzed by agarose gel electrophoresis to check the length of theamplified fragment.

[0153] FLINT cDNA generated in this manner is used as a template forintroducing appropriate point mutations (i.e. construction of FLINTanalog cDNAs). A suitable protocol is described in detail in “CurrentProtocols in Molecular Biology”, volume 1, section 8.5.7 (John Wiley andSons, Inc. publishers). Briefly, synthetic oligonucleotides are designedto incorporate one or more point mutation(s) at one end of an amplifiedfragment, e.g. at position 218 of SEQ ID NO:1. Following first strandPCR, the amplified fragments encompassing the mutation are annealed witheach other and extended by mutually primed synthesis. Annealing isfollowed by a second PCR step utilizing 5′ forward and 3′ reverse endprimers in which the entire mutagenized fragment gets amplified and isready for subcloning into the appropriate vector.

[0154] The skilled artisan understands that the degeneracy of thegenetic code provides multiple codons in some instances for a givenamino acid. All such nucleic acid sequence variants are intended to bewithin the scope of the invention.

[0155] Nucleic acid molecules of the present invention can be in theform of RNA, such as mRNA, hnRNA, or any other form, or in the form ofDNA, including, but not limited to, cDNA and genomic DNA obtained bycloning or produced synthetically, or any combination thereof. The DNAcan be triple-stranded, double-stranded or single-stranded, or anycombination thereof. Any portion of at least one strand of the DNA orRNA can be the coding strand, also known as the sense strand, or it canbe the non-coding strand, also referred to as the anti-sense strand.

[0156] “Host cell” refers to any eucaryotic, procaryotic, or other cellor pseudo cell or membrane-containing construct that is suitable forpropagating and/or expressing an isolated nucleic acid that isintroduced into a host cell by any suitable means known in the art(e.g., transformation or transfection, or the like), or induced toexpress an endogenous polydeoxynucleic acid. The cell can be part of atissue or organism, isolated in culture or in any other suitable form.

[0157] A variety of eucaryotic expression systems such as yeast, insectcell lines, plant and mammalian cells, are known to those of skill inthe art.

[0158] Nucleic acid sequences encoding a FLINT polypeptide of thepresent invention can be ligated to various expression vectors for usein transfecting cell cultures of, for instance, mammalian, insect, orplant origin. Preferred cell cultures useful for the production of FLINTpolypeptides are mammalian cells. Mammalian cell monolayers orsuspensions may be used. A number of suitable mammalian host cell lineshave been developed in the art, including the AV12, 293 EBNA, HEK293,BHK21, and CHO cell lines. Expression vectors for these cell lines caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer, and processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites (e.g., bovine growth hormone poly A additionsite), and transcriptional terminator sequences.

[0159] Expression of FLINT in Host Cells

[0160] Briefly, the expression of isolated nucleic acids encoding aFLINT analog of the present invention will typically be achieved byoperably linking a DNA or cDNA encoding an analog to a promoter (whichis either constitutive or inducible), followed by incorporation into anexpression vector. The vectors can be suitable for replication andintegration in either procaryotes or eucaryotes. To obtain high levelexpression of a cloned gene, it is desirable to construct expressionvectors that contain a promoter to direct transcription, a ribosomebinding site for translational initiation, and atranscription/translation terminator. One of skill in the art wouldrecognize that modifications can be made to a protein of the presentinvention without diminishing its biological activity. For example, somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification handle sequences.

[0161] Protein Purification

[0162] A FLINT polypeptide of the present invention comprising N-linked,and/or O-linked, an/or N/O-linked oligosaccharides can be recovered andpurified from recombinant cells that express said polypeptide by anysuitable method, well-known to the skilled artisan including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, and immobilized metal ion affinity chelatingchromatography, “IMAC,” as taught in U.S. Pat. No. 4,569,974 hereinincorporated by reference, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Preferably, species of FLINTpolypeptides of the present invention are separated and purified byreverse phase high performance liquid chromatography (“RP-HPLC”).

[0163] Therapeutic Applications

[0164] FLINT polypeptides inhibit the binding of Fas to FasL and LIGHTto LTβR and TR2/HVEM receptors, and can be used to treat or prevent adisease and/or condition that may be associated with such bindinginteractions.

[0165] FLINT polypeptides of the present invention are clinically and/ortherapeutically useful for treating and/or preventing a plurality ofdiseases including Rheumatoid arthritis (Elliott et al., Lancet344:1105-10 (1994)), fibroproliferative lung disease, fibrotic lungdisease, acute lung injury, acute respiratory distress syndrome, HIV(Dockrell et al., J. Clin. Invest. 101:2394-2405 (1998)), Ischemia(Sakurai et al. 1998 Brain Res 797:23-28), Brain trauma/injury (Ertel etal. 1997 J Neuroimmunol 80:93-6), chronic renal failure (Schelling etal. 1998 Lab Invest 78:813-824), Graft-vs-Host Disease (GVHD) (Hattoriet al. 1998 Blood 11:4051-4055), Cutaneous inflammation (Orteu at al.1998 J Immunol 161:1619-1629), Vascular leak syndrome (Rafi et al. 1998J Immunol 161:3077-3086), Helicobacter pylori infection (Rudi et al.1998 J Clin Invest 102:1506-1514), Goiter (Tamura et al. 1998Endocrinology 139:3646-3653), Atherosclerosis (Sata and Walsh, 1998 JClin Invest 102:1682-1689), IDDM (Itoh et al. 1997. J Exp Med186:613-618), Osteoporosis (Jilka et al. 1998 J Bone Min Res13:793-802), inflammatory bowel disease, ulcerative colitis, Crohn'sDisease (van Dullemen et al. 1995 Gastroenterology 109:129-35), organpreservation and transplant (graft) rejection (Lau et al. 1996 Science273:109-112), Sepsis (Faist and Kim. 1998 New Horizons 6:S97-102),Pancreatitis (Neoptolemos et al. 1998 Gut 42:886-91), Cancer (melanoma,colon and esophageal) (Bennett et al. 1998 J Immunol 160:5669-5675),Autoimmune disease (IBD, psoriasis, Down's Syndrome (Seidi et al.,Neuroscience Lett. 260:9 (1999), multiple sclerosis (D'Souza et al. 1996J Exp Med 184:2361-70), and chronic obstructive pulmonary disease.

[0166] An “effective amount” of a FLINT polypeptide is an amount whichachieves a desired therapeutic or prophylactic effect in a subject witha disease, or in a method of the invention, that may be associated withaberrant Fas/Fas Ligand binding and/or LIGHT mediated binding. Oneexample of such a process is runaway apoptosis. Alternatively, an“effective amount” of a FLINT polypeptide is a quantity sufficient toachieve a desired therapeutic and/or prophylactic effect in a subjectwith inflammation caused by Fas Ligand induced neutrophil activation orany of the other aforementioned diseases associated with aberrant FasLigand activity.

[0167] The amount of FLINT administered to the individual will depend onthe type and severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. It will also depend on the degree, severity and type ofdisease. The skilled artisan will be able to determine appropriatedosages depending on these and other factors.

[0168] As a general proposition, the total pharmaceutically effectiveamount of FLINT of the present invention administered parenterally perdose will be in the range of about 1 μg/kg/day to 10 mg/kg/day ofpatient body weight, particularly 2 mg/kg/day to 8 mg/kg/day, moreparticularly 2 mg/kg/day to 4 mg/kg/day, even more particularly 2.2mg/kg/day to 3.3 mg/kg/day, and finally 2.5 mg/kg/day, although, asnoted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day. If given continuously,the FLINT of the present invention are typically administered at a doserate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4injections per day or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed. Thelength of treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

[0169] Pharmaceutical compositions containing a FLINT polypeptide of thepresent invention may be administered orally, rectally, intracranially,parenterally, intracisternally, intravaginally, intraperitoneally,topically (as by powders, ointments, drops or transdermal patch),transdermally, intrathecally, bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinincludes, but is not limited to, modes of administration which includeintravenous, intramuscular, intraperitoneal, intrasternal, subcutaneousand intraarticular injection, infusion and implants comprising FLINT.

[0170] For parenteral administration, the FLINT polypeptides of thepresent invention are formulated generally by mixing at the desireddegree of purity, in a unit dosage injectable form (solution,suspension, or emulsion), with a pharmaceutically acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation. For example, the formulation preferably does not includeoxidizing agents and other compounds that are known to be deleterious topolypeptides.

[0171] Generally, the formulations are prepared by contacting the FLINTof the present invention uniformly and intimately with liquid carriersor finely divided solid carriers or both. Then, if necessary, theproduct is shaped into the desired formulation. Preferably the carrieris a parenteral carrier, more preferably a solution that is isotonicwith the blood of the recipient. Examples of such carrier vehiclesinclude water, saline, Ringer's solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein, as well as liposomes.

[0172] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid: or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0173] The FLINT polypeptides of the present invention are typicallyformulated in such vehicles at a concentration of about 0.1 mg/ml to 100mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will beunderstood that the use of certain of the foregoing excipients,carriers, or stabilizers will result in the formation of salts of theFLINT analogs of the present invention.

[0174] The invention is illustrated by the following examples which arenot intended to be limiting in any way.

EXAMPLE 1 Production of a Vector for Expressing FLINT Analog R218Q

[0175] FLINT variant R218Q was constructed by mutagenic PCR startingfrom a wild-type FLINT template. See e.g. Saiki R. K. et al. Science239:487-491 (1988), and “Current Protocols in Molecular Biology”, Vol 1,section 8.5.7 (John Wiley and Sons, Inc. publishers). The R218Q mutantsubstitutes an arginine residue found at amino acid 218 with glutamine.The mutagenic PCR process utilized a SOEing reaction (i.e. StrandOverlap Extension) to create specific mutations in the native FLINTtemplate for the purpose of changing the amino acid sequence at position218, and further for introducing restriction enzyme tags foridentification purposes. Generally, SOEing reactions require the use offour primers, two in the forward orientation (termed A, SEQ ID NO:5, andC, SEQ ID NO:7) and two in the reverse orientation (termed B, SEQ IDNO:6 and D, SEQ ID NO:8). The SOEing reaction amplifies a nucleic acidsequence (e.g. gene sequence) in two stages. The first step is toamplify “half” the gene by performing an A to B reaction followed by aseparate C to D reaction. In constructing the R218Q mutant, the B and Cprimers were targeted to the same area of the gene but on oppositestrands. Mismatch priming from both oligonucleotide primers institutesthe mutation. After these two reactions were completed, the productswere isolated and mixed for use as the template for the A to D reaction,which yields the desired mutated product. The primers involved in thecloning of R218Q were: Primer A: CF 107 (39 nt)GCACCAGGGTACCAGGAGCTGAGGAGTGTGAGCGTGCCG Primer B: CF 111 (44 nt)TCAGCTGCAAGGCGGCGCGCCCCGCTTGTGGTGTCGGACCCCAG Primer C: CF 112 (44 nt)GGGGTCCGACACCACAAGCGGGGCGCGCCGCCTTGCAGCTGAAG Primer D: CF 110 (43 nt)GCACAGAATTCATCAGTGCACAGGGAGGAAGCGCTCACGGACG

[0176] Using the forward primer C as a reference, the bold G and C showthe silent changes necessary to introduced an AscI site. Thisrecognition site is underlined in primers B and C.

[0177] The 311 base pair amplified fragment carrying the R218Q mutationwas sub-cloned using a 5′ KpnI site (GGTACC) and a 3′ EcoRI site(GAATTC). The native FLINT sequence has a naturally occurring internalKpnI site around amino acid position 176. The EcoRI site was introducedfor sub-cloning purposes and lies downstream of the stop codons. Thesesites are underlined in primers A and D respectively. The 311 bpfragment was incorporated into the full length FLINT sequence. This wasaccomplished through the following steps:

[0178] The 311 bp fragment was placed into an intermediate vector,pCR2.1- TOPO, which utilizes the adenine overhangs established after PCRfor ligation.

[0179] Once incorporated, a KpnI to EcoRI digestion removed a 289 bpfragment. (Note: the size of the PCR fragment decreased from 311 bp to289 bp due to the digestion). The mutated fragment was used to replacethe corresponding segment in the wild type FLINT gene by directionalligation.

[0180] FLINT/pJB03 was digested with KpnI to EcoRI to produce twofragments

[0181] Fragment 1: 6070 bp

[0182] Fragment 2: 289 bp

[0183] The 6070 bp fragment carrying the FLINT gene was isolated andligated with the 289 bp PCR product removed from the pCR 2.1-TOPO vectorto create R218Q/pJB03. Positive clones were identified by restrictiondigestion and subsequently confirmed by sequence analysis. The R218Qanalog contained in R218Q/pJB03 was shuffled into the pIG3 vector bymeans of a NheI to XbaI undirected ligation. R218Q/pJB03 was digestedwith NheI to XbaI to yield fragments of 932 and 5427 bp. The 932 bpFLINT R218Q containing fragment was isolated. The 932 bp NheI and XbaIfragment of R218Q was ligated with the 8510 bp linearized pIC 3 vectorto generate clones in both forward and inverse orientations.

EXAMPLE 2 Construction of Vector pIG3 for Expression of FLINT inMammalian Cells

[0184] A bicistronic expression vector was constructed by inserting intomammalian expression vector pGTD (Gerlitz, B. et al., 1993, BiochemicalJournal 295:131) a PCR fragment encoding an “internal ribosome entrysite”/enhanced green fluorescent polypeptide (IRES/eGFP). The newvector, designated pIG3, contains the following sequence landmarks: theEla-responsive GBMT promoter (D. T. Berg et al., 1993 BioTechniques14:972; D. T. Berg et al., 1992 Nucleic Acids Research 20:5485); amultiple cloning site (MCS); the IRES sequence from encephalomyocarditisvirus (EMCV); the eGFP coding sequence (Cormack, et al., 1996 Gene173:33, Clontech); the SV40 small “t” antigen splicesite/polyadenylation sequences; the SV40 early promoter and origin ofreplication; the murine dihydrofolate reductase (dhfr) coding sequence;and the ampicillin resistance gene and origin of replication frompBR322.

[0185] Based on the human FLINT cDNA sequence (e.g. SEQ ID NO:3),forward and reverse PCR primers were synthesized bearing BclIrestriction sites at their respective 5′ends. These primers were used toamplify the FLINT cDNA. The FLINT cDNA orientation and nucleotidesequence was confirmed by restriction digest and double strandedsequencing of the insert. The approximately 900 base pair amplifiedFLINT analog PCR product was digested with restriction endonucleasesNheI and XbaI, respectively, to generate a fragment bearing NheI andXbaI sticky ends. This fragment was subsequently ligated into a uniqueXbaI site of pIG3 to generate recombinant plasmid pIG3-FLINT. The insertencoding FLINT can be modified at the C-terminus of the analog tointroduce a cleavable hexahistidine (His6) cassette to facilitate analogpurification.

EXAMPLE 3 Isolation of High-Producing FLINT Clone from AV12 RGT18Transfectants

[0186] The recombinant plasmid of Example 2 carries the FLINT gene andencodes resistance to methotrexate. In addition, the construct containsa gene encoding a fluorescent polypeptide, GFP, on the same transcriptand immediately 3′ to the FLINT gene. Since high level expression of GFPwould require a high level of expression of the FLINT-GFP mRNA, highlyfluorescent clones have a greater probability of producing high levelsof FLINT.

[0187] AV12 RGT18 cells are transfected using a calcium phosphateprocedure with recombinant plasmid pIG1. Cells resistant to 250 nMmethotrexate are selected and pooled. The pool of resistant clones issubjected to fluorescence assisted cell sorting (FACS), and cells havingfluorescence values in the top 5% of the population are sorted into apool and as single-cells. High fluorescence pools are subjected to twosuccessive sorting cycles. Pools and individual clones from the firstand second cycles are analyzed for FLINT production by ELISA. Pools orclones expressing FLINT at the highest level are used for scale-up andFLINT purification.

EXAMPLE 4 Large Scale Purification of Native O-linked FLINT Polypeptide

[0188] Large scale production of mature FLINT (mFLINT) was carried outby growing stable clones of AV12 RGT 18 cells transfected withpIG3-FLINT in several 10 liter spinners. After reaching confluency,cells were further incubated for 2-3 more days to secrete maximum amountof mFLINT into the growth medium. Medium containing mFLINT was adjustedto 0.1% CHAPS and concentrated in an Amicon ProFlux M12 tangentialfiltration system to 350 ml. The concentrated media was adjusted to pH6.0 and passed over a SP Sepharose Fast Flow (Pharmacia, 500 ml) at aflow rate of 7 ml/min. The column was washed with buffer A (20 mM MOPS,0.1% CHAPS, pH 6.0) until the absorbance (280 nm) returned to baselineand bound polypeptides were eluted with a linear gradient from 0 to 1 MNaCl (in buffer A) developed over four column volumes. Fractionscontaining mFLINT were pooled and passed over Vydac C4 column (100 ml)equilibrated with 0.1% TFA/H₂0 at a flow rate of 10 ml/min.

[0189] The bound mFLINT was separated into a predominantly N-linkedspecies, and a predominantly N/O-linked mFLINT species by elution with alinear gradient 0% to 50% CH₃CN/0.1% TFA over 10 column volumes followedby a linear gradient from 50% to 90% CH₃CN/0.1% TFA over 1 columnvolumne. Fractions containing N/O-linked mFLINT and N-linked mFLINT wereseparately pooled, concentrated under vacuum to approximately 4 ml andan equal volume of PBS, 0.5 M NaCl, 10% glycerol, 100 mM Tris, 50 mMEDTA, pH 7.0 was added. The purified samples were dialyzed against fourliters of PBS, 0.5 M NaCl, 10% glycerol, 1 mM EDTA, pH 7.4 three timesand four liters of PBS, 0.5 M NaCl, 10% glycerol, pH 7.4 three times.Substantially pure fractions obtained in this fashion contain eitherpredominantly N/O-linked or N-linked mFLINT. Analysis by SDS-PAGErevealed these fractions to be greater than 85% pure. The N-terminalsequence of mFLINT was confirmed on the purified polypeptides. LC-MS wasperformed on the polypeptides to confirm the location of the glycans.

EXAMPLE 5 Quantitation of FLINT Analogs

[0190] FLINT analogs can be quantitated in crude media of transfectedcells and during purification procedure by a developed FLINT ELISA.ELISA uses anti-FLINT polyclonal antibody TKD-028(1494) as a captureantibody and biotinylated anti-FLINT TKD-076A as a primary antibody in a“sandwich” assay. ELISA is developed by streptavidin derivatized horseradish peroxidase (SA-HRP) using OPD as a substrate and monitoring theabsorbance at 490 mn. The useful range of such an ELISA is from 0.2-20ng/ml.

EXAMPLE 6 Effect of ZnCl₂ on Thermal Stability of FLINT

[0191] The conformational stability of FLINT in the presence or absenceof ZnCl₂ was monitored by differential scanning calorimetry (DSC). Datawas collected on a VP-DSC MicroCalorimeter using VPViewer software andOrigin DSC software for data analysis. The matched sample and referencecells had a working volume of 0.5 mL. FLINT samples were dialyzedagainst buffer containing 25 mM Tris, 150 mM NaCl at pH 6.8 overnightand the concentration of protein was determined by UV absorbance at 277nm using extinction coefficient of 0.786 for 1 mg/mL protein sample in1-cm pathlength cell.. Buffer was also run overnight in both cells toestablish a thermal history prior to sample runs. Proteins were thendiluted to approximately 0.15 mg/mL, and the dialysate was used as thereference solution. 2 ul or 4 ul of ZnCl₂ at 5 mM was added to 1 mLprotein sample to obtain 10 uM ZnCl₂ concentration. After degassing,sample and reference were loaded in cells with 2.5 mL needle through afilling funnel. Pressure was kept at approximately 30 psi. with apressure cap. Data was collected between 5° and 100° C. The denaturationprocess was partially reversible. The material was recovered andanalyzed by RP-HPLC to determine the ratio of N and O-linkedglycosylation species. The fraction of N/O-linked species increased from49% before thermal denaturation to 55% after denaturation, indicatingthat material containing O-linked glycosylation is more stable thanmaterial containing only N-linked glycosylation.

EXAMPLE 7 Large Scale N and O-linked FLINT Analog PolypeptidePurification

[0192] Large scale production of FLINT R218Q is carried out by firstgrowing stable pIG1-R218Q-containing AV12 RGT 18 cells in several 10liter spinners. After reaching confluency, cells are further incubatedfor 2-3 more days to secrete maximum amount of FLINT analog into media.Medium containing FLINT analog is adjusted to 0.1% CHAPS andconcentrated in an Amicon ProFlux M12 tangential filtration system to350 ml. The concentrated medium is adjusted to pH 6.0 and passed over aSP Sepharose Fast Flow (Pharmacia, 500 ml) at a flow rate of 7 ml/min.The column is washed with buffer A (20 mM MOPS, 0.1% CHAPS, pH 6.0)until the absorbance (280 nm) returns to baseline and the boundpolypeptides are eluted with a linear gradient from 0 to 1 M NaCl (inbuffer A) developed over four column volumes. Fractions containing FLINTare pooled and passed over Vydac C4 column (100 ml) equilibrated with0.1% TFA/H₂O at a flow rate of 10 ml/min. The bound FLINT analog isseparated into N-linked and O-linked FLINT analog and N-linked FLINTanalog by elution with a linear gradient 0% to 50% CH₃CN/0.1% TFA over10 column volumes followed by a linear gradient from 50% to 90%CH₃CN/0.1% TFA over 1 column volume. Fractions containing N- andO-linked FLINT and N-linked FLINT are pooled separately, concentratedunder vacuum to approximately 4 ml and an equal volume of PBS, 0.5 MNaCl, 10% glycerol, 100 mM Tris, 50 mM EDTA, pH 7.0 is added. Thepurified samples are dialyzed against four liters of PBS, 0.5 M NaCl,10% glycerol, 1 mM EDTA, pH 7.4 three times and four liters of PBS, 0.5M NaCl, 10% glycerol, pH 7.4 three times. Fractions containing N- andO-linked versus N-linked FLINT are analyzed by SDS-PAGE. The N-terminalsequence of FLINT is confirmed on the purified polypeptides. LC-MS isperformed on the polypeptides to confirm the location of the glycans.

EXAMPLE 8 Use of O-linked Glycosylated FLINT to Treat ALI Patient

[0193] A 55 year-old male presents to the emergency departmentunconscious. His family states that he was being treated as anoutpatient for bronchitis for the past few days but worsened despiteantibiotics. He has no relevant past history and his only medication wasa third generation oral cephalosporin. Physical examination reveals anobtunded, cyanotic male who is hypotensive, tachypneic, and tachycardic,and who has bilateral lung congestion consistent with pulmonary edema.There is no evidence of congestive heart failure. Tests reveal hypoxemia(based on PaO2/FiO2) and bilateral lung infiltrates withoutcardiomegaly, consistent with a diagnosis of acute lung injury. Based onthe history it is concluded that the lung injury was a direct result ofcommunity-acquired pneumonia, and that the patient met the hypoxemiacriteria for ALI within the last 12 hours. Ventilation measures includeuse of PEEP and low tidal volume. As soon as adequate oxygenation isconfirmed, treatment with O-linked glycosylated FLINT is initiated inthe ER as an iv bolus of 2.5 mg/kg, followed by a continuous infusion of0.1 mg/minute. FLINT along with aggressive supportive measures (e.g.,positive ventilation, intravenous fluids, pressors, and nutritionalsupport) are continued for four days in the ICU, at which time the FLINTis discontinued. Over the following 3 days, the patient begins torecover and is extubated on Day 8. He has an uneventful recovery and 6months following discharge has no evidence of residual lung disease byblood gas and spirometry.

EXAMPLE 9 Effect of ZnCl₂ on Thermal Stability of FLINT Analog

[0194] The conformational stability of FLINT analog R218Q in thepresence or absence of ZnCl₂ is monitored by differential scanningcalorimetry (DSC). Data is collected on a VP-DSC MicroCalorimeter usingVPViewer software and Origin DSC software for data analysis. The matchedsample and reference cells have a working volume of 0.5 mL. FLINTsamples are dialyzed against buffer containing 25 mM Tris, 150 mM NaClat pH 6.8 overnight and the concentration of protein determined by UVabsorbance at 277 nm using extinction coefficient of 0.786 for 1 mg/mLprotein sample in 1 cm path length cell. Buffer is also run overnight inboth cells to establish a thermal history prior to sample runs. Proteinsare diluted to approximately 0.15 mg/mL, and the dialysate is used asthe reference solution. 2 ul or 4 ul of ZnCl₂ at 5 mM is added to 1 mLprotein sample to obtain 10 uM and 20 uM ZnCl₂ concentration,respectively. After degassing, sample and reference are loaded in cellswith 2.5 mL needle through a filling funnel. Pressure is kept atapproximately 30 psi. with a pressure cap. Data is collected between 5°and 100° C. Thermal scan of FLINT analog with or without ZnCl₂ is taken.Data is analyzed to obtain the midpoint of thermal transition (T_(m)).FLINT analog sample contains two populations of molecules, onecontaining only N-linked glycosylation at Asn144, the other containingboth N-linked glycosylation and O-linked glycosylation at T174 and/orT216. These two populations of molecules are separated by RP-HPLC. Thedata suggest that ZnCl₂ has a stabilizing effect on FLINT analogcontaining additional O-linked glycosylation compared to FLINT onlycontaining N-linked glycosylation.

EXAMPLE 10 Production of the FLINT Metabolite

[0195] FLINT was purified from AV12 RGT 18 cells transfected with arecombinant vector carrying a FLINT cDNA (SEQ ID NO:1 or SEQ ID NO:3).This material was cleaved with thrombin at a weight ratio of 1 to 100(thrombin to FLINT) for three hours at room temperature, dialyzedagainst 20 mM MOPS, 0.1% CHAPS, pH 6.5, and passed over a SP Sepharosecolumn at a flow rate of 1 ml/min. The column was washed with buffer A(20 mM MOPS, 0.1% CHAPS, pH 6.5) until the absorbance returned tobaseline. The bound metabolite (amino acids 1 to 218 of SEQ ID NO:1) waseluted with a linear gradient from 0 to 300 mM NaCl (in buffer A)developed over 10 min. followed by a linear gradient for 0.3 to 0.5 M(in buffer A). Fractions were analyzed by SDS-PAGE and massspectrometry. Fractions containing only the FLINT metabolite (1-218)were pooled and concentrated in Millipore Ultrafree centrifugal filter.The concentrated metabolite (1-218) was again analyzed by SDS-PAGE andmass spectrometry to assess purity. N-terminal sequencing confirmed theidentity of the purified material as FLINT metabolite (1-218).

EXAMPLE 11 Improving Stability of FLINT Metabolite Through O-linkedGlycosylation

[0196] When FLINT was injected subcutaneously or intravenously in mice,a metabolite containing amino acids 1-218 of FLINT was formed. Thismetabolite can also be generated using thrombin cleavage in vitro.

[0197] To investigate the effect of differential N and O-linkedglycosylation on the stability of FLINT metabolite, FLINT produced as inExamples 2 through 4 was treated with thrombin and the metaboliteproduced thereby analyzed by reverse phase HPLC and differentialscanning calorimetry As shown in FIG. 4, FLINT metabolite contains twospecies when analyzed by RP-HPLC, N-linked and N/O-linked. Metabolitecontaining N/O-linked glycosylation is more stable than N-linkedspecies, as shown by RP-HPLC. As the sample was concentrated in about200 mM NaCl, 20 mM MOPS buffer using Millipore ultrafree-4 centrifugalfilter unit (10,000 MWCO membrane), material containing only N-linkedglycosylation was preferentially lost compared to material containingboth N-linked and O-linked glycosylation, leading to the change in therelative peak areas for these two species (FIG. 5).

[0198] The thermal stability of FLINT metabolite in PBS monitored bydifferential scanning calorimetry showed two transitions as shown inFIG. 6, with midpoint of transition temperature at 48° C. for the firsttransition and 57° C. for the second transition. The high meltingtemperature transition may be attributed to FLINT metabolite containingboth N- and O-linked glycosylation. The second transition disappearedwith addition of 2 mM EDTA into the sample, suggesting that divalentmetal ions are likely to be involved in the stabilization of FLINTmetabolite.

[0199] The data presented above suggest that additional O-linkedglycosylation provides better physical stability to FLINT metabolite. Itis conceivable that mutations to introduce additional glycosylationsites or fusion protein of FLINT metabolite with Fc can also bebeneficial to the stability of FLINT metabolite.

1 9 1 271 PRT Homo sapiens 1 Val Ala Glu Thr Pro Thr Tyr Pro Trp Arg AspAla Glu Thr Gly Glu 1 5 10 15 Arg Leu Val Cys Ala Gln Cys Pro Pro GlyThr Phe Val Gln Arg Pro 20 25 30 Cys Arg Arg Asp Ser Pro Thr Thr Cys GlyPro Cys Pro Pro Arg His 35 40 45 Tyr Thr Gln Phe Trp Asn Tyr Leu Glu ArgCys Arg Tyr Cys Asn Val 50 55 60 Leu Cys Gly Glu Arg Glu Glu Glu Ala ArgAla Cys His Ala Thr His 65 70 75 80 Asn Arg Ala Cys Arg Cys Arg Thr GlyPhe Phe Ala His Ala Gly Phe 85 90 95 Cys Leu Glu His Ala Ser Cys Pro ProGly Ala Gly Val Ile Ala Pro 100 105 110 Gly Thr Pro Ser Gln Asn Thr GlnCys Gln Pro Cys Pro Pro Gly Thr 115 120 125 Phe Ser Ala Ser Ser Ser SerSer Glu Gln Cys Gln Pro His Arg Asn 130 135 140 Cys Thr Ala Leu Gly LeuAla Leu Asn Val Pro Gly Ser Ser Ser His 145 150 155 160 Asp Thr Leu CysThr Ser Cys Thr Gly Phe Pro Leu Ser Thr Arg Val 165 170 175 Pro Gly AlaGlu Glu Cys Glu Arg Ala Val Ile Asp Phe Val Ala Phe 180 185 190 Gln AspIle Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu 195 200 205 AlaPro Glu Gly Trp Gly Pro Thr Pro Arg Ala Gly Arg Ala Ala Leu 210 215 220Gln Leu Lys Leu Arg Arg Arg Leu Thr Glu Leu Leu Gly Ala Gln Asp 225 230235 240 Gly Ala Leu Leu Val Arg Leu Leu Gln Ala Leu Arg Val Ala Arg Met245 250 255 Pro Gly Leu Glu Arg Ser Val Arg Glu Arg Phe Leu Pro Val His260 265 270 2 813 DNA Homo sapiens 2 gtggcagaaa cacccaccta cccctggcgggacgcagaga caggggagcg gctggtgtgc 60 gcccagtgcc ccccaggcac ctttgtgcagcggccgtgcc gccgagacag ccccacgacg 120 tgtggcccgt gtccaccgcg ccactacacgcagttctgga actacctgga gcgctgccgc 180 tactgcaacg tcctctgcgg ggagcgtgaggaggaggcac gggcttgcca cgccacccac 240 aaccgtgcct gccgctgccg caccggcttcttcgcgcacg ctggtttctg cttggagcac 300 gcatcgtgtc cacctggtgc cggcgtgattgccccgggca cccccagcca gaacacgcag 360 tgccagccgt gccccccagg caccttctcagccagcagct ccagctcaga gcagtgccag 420 ccccaccgca actgcacggc cctgggcctggccctcaatg tgccaggctc ttcctcccat 480 gacaccctgt gcaccagctg cactggcttccccctcagca ccagggtacc aggagctgag 540 gagtgtgagc gtgccgtcat cgactttgtggctttccagg acatctccat caagaggctg 600 cagcggctgc tgcaggccct cgaggccccggagggctggg gtccgacacc aagggcgggc 660 cgcgcggcct tgcagctgaa gctgcgtcggcggctcacgg agctcctggg ggcgcaggac 720 ggggcgctgc tggtgcggct gctgcaggcgctgcgcgtgg ccaggatgcc cgggctggag 780 cggagcgtcc gtgagcgctt cctccctgtgcac 813 3 300 PRT Homo sapiens 3 Met Arg Ala Leu Glu Gly Pro Gly Leu SerLeu Leu Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu Pro Val ProAla Val Arg Gly Val Ala Glu 20 25 30 Thr Pro Thr Tyr Pro Trp Arg Asp AlaGlu Thr Gly Glu Arg Leu Val 35 40 45 Cys Ala Gln Cys Pro Pro Gly Thr PheVal Gln Arg Pro Cys Arg Arg 50 55 60 Asp Ser Pro Thr Thr Cys Gly Pro CysPro Pro Arg His Tyr Thr Gln 65 70 75 80 Phe Trp Asn Tyr Leu Glu Arg CysArg Tyr Cys Asn Val Leu Cys Gly 85 90 95 Glu Arg Glu Glu Glu Ala Arg AlaCys His Ala Thr His Asn Arg Ala 100 105 110 Cys Arg Cys Arg Thr Gly PhePhe Ala His Ala Gly Phe Cys Leu Glu 115 120 125 His Ala Ser Cys Pro ProGly Ala Gly Val Ile Ala Pro Gly Thr Pro 130 135 140 Ser Gln Asn Thr GlnCys Gln Pro Cys Pro Pro Gly Thr Phe Ser Ala 145 150 155 160 Ser Ser SerSer Ser Glu Gln Cys Gln Pro His Arg Asn Cys Thr Ala 165 170 175 Leu GlyLeu Ala Leu Asn Val Pro Gly Ser Ser Ser His Asp Thr Leu 180 185 190 CysThr Ser Cys Thr Gly Phe Pro Leu Ser Thr Arg Val Pro Gly Ala 195 200 205Glu Glu Cys Glu Arg Ala Val Ile Asp Phe Val Ala Phe Gln Asp Ile 210 215220 Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu Ala Pro Glu 225230 235 240 Gly Trp Gly Pro Thr Pro Arg Ala Gly Arg Ala Ala Leu Gln LeuLys 245 250 255 Leu Arg Arg Arg Leu Thr Glu Leu Leu Gly Ala Gln Asp GlyAla Leu 260 265 270 Leu Val Arg Leu Leu Gln Ala Leu Arg Val Ala Arg MetPro Gly Leu 275 280 285 Glu Arg Ser Val Arg Glu Arg Phe Leu Pro Val His290 295 300 4 29 PRT Homo sapiens 4 Met Arg Ala Leu Glu Gly Pro Gly LeuSer Leu Leu Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu Pro ValPro Ala Val Arg Gly 20 25 5 39 DNA Artificial Sequence Description ofArtificial Sequence oligo primer 5 gcaccagggt accaggagct gaggagtgtgagcgtgccg 39 6 44 DNA Artificial Sequence Description of ArtificialSequence oligo primer 6 tcagctgcaa ggcggcgcgc cccgcttgtg gtgtcggacc ccag44 7 44 DNA Artificial Sequence Description of Artificial Sequence oligoprimer 7 ggggtccgac accacaagcg gggcgcgccg ccttgcagct gaag 44 8 43 DNAArtificial Sequence Description of Artificial Sequence oligo primer 8gcacagaatt catcagtgca cagggaggaa gcgctcacgg acg 43 9 936 DNA Homosapiens CDS (25)..(924) 9 gctctccctg ctccagcaag gacc atg agg gcg ctg gagggg cca ggc ctg 51 Met Arg Ala Leu Glu Gly Pro Gly Leu 1 5 tcg ctg ctgtgc ctg gtg ttg gcg ctg cct gcc ctg ctg ccg gtg ccg 99 Ser Leu Leu CysLeu Val Leu Ala Leu Pro Ala Leu Leu Pro Val Pro 10 15 20 25 gct gta cgcgga gtg gca gaa aca ccc acc tac ccc tgg cgg gac gca 147 Ala Val Arg GlyVal Ala Glu Thr Pro Thr Tyr Pro Trp Arg Asp Ala 30 35 40 gag aca ggg gagcgg ctg gtg tgc gcc cag tgc ccc cca ggc acc ttt 195 Glu Thr Gly Glu ArgLeu Val Cys Ala Gln Cys Pro Pro Gly Thr Phe 45 50 55 gtg cag cgg ccg tgccgc cga gac agc ccc acg acg tgt ggc ccg tgt 243 Val Gln Arg Pro Cys ArgArg Asp Ser Pro Thr Thr Cys Gly Pro Cys 60 65 70 cca ccg cgc cac tac acgcag ttc tgg aac tac ctg gag cgc tgc cgc 291 Pro Pro Arg His Tyr Thr GlnPhe Trp Asn Tyr Leu Glu Arg Cys Arg 75 80 85 tac tgc aac gtc ctc tgc ggggag cgt gag gag gag gca cgg gct tgc 339 Tyr Cys Asn Val Leu Cys Gly GluArg Glu Glu Glu Ala Arg Ala Cys 90 95 100 105 cac gcc acc cac aac cgtgcc tgc cgc tgc cgc acc ggc ttc ttc gcg 387 His Ala Thr His Asn Arg AlaCys Arg Cys Arg Thr Gly Phe Phe Ala 110 115 120 cac gct ggt ttc tgc ttggag cac gca tcg tgt cca cct ggt gcc ggc 435 His Ala Gly Phe Cys Leu GluHis Ala Ser Cys Pro Pro Gly Ala Gly 125 130 135 gtg att gcc ccg ggc accccc agc cag aac acg cag tgc cag ccg tgc 483 Val Ile Ala Pro Gly Thr ProSer Gln Asn Thr Gln Cys Gln Pro Cys 140 145 150 ccc cca ggc acc ttc tcagcc agc agc tcc agc tca gag cag tgc cag 531 Pro Pro Gly Thr Phe Ser AlaSer Ser Ser Ser Ser Glu Gln Cys Gln 155 160 165 ccc cac cgc aac tgc acggcc ctg ggc ctg gcc ctc att gtg cca ggc 579 Pro His Arg Asn Cys Thr AlaLeu Gly Leu Ala Leu Ile Val Pro Gly 170 175 180 185 tct tcc tcc cat gacacc ctg tgc acc agc tgc act ggc ttc ccc ctc 627 Ser Ser Ser His Asp ThrLeu Cys Thr Ser Cys Thr Gly Phe Pro Leu 190 195 200 agc acc agg gta ccagga gct gag gag tgt gag cgt gcc gtc atc gac 675 Ser Thr Arg Val Pro GlyAla Glu Glu Cys Glu Arg Ala Val Ile Asp 205 210 215 ttt gtg gct ttc caggac atc tcc atc aag agg ctg cag cgg ctg ctg 723 Phe Val Ala Phe Gln AspIle Ser Ile Lys Arg Leu Gln Arg Leu Leu 220 225 230 cag gcc ctc gag gccccg gag ggc tgg gct ccg aca cca agg gcg ggc 771 Gln Ala Leu Glu Ala ProGlu Gly Trp Ala Pro Thr Pro Arg Ala Gly 235 240 245 cgc gcg gcc ttg cagctg aag ctg cgt cgg cgg ctc acg gag ctc ctg 819 Arg Ala Ala Leu Gln LeuLys Leu Arg Arg Arg Leu Thr Glu Leu Leu 250 255 260 265 ggg gcg cag gacggg gcg ctg ctg gtg cgg ctg ctg cag gcg ctg cgc 867 Gly Ala Gln Asp GlyAla Leu Leu Val Arg Leu Leu Gln Ala Leu Arg 270 275 280 gtg gcc agg atgccc ggg ctg gag cgg agc gtc cgt gag cgc ttc ctc 915 Val Ala Arg Met ProGly Leu Glu Arg Ser Val Arg Glu Arg Phe Leu 285 290 295 cct gtg cactgatcctggc cc 936 Pro Val His 300

What is claimed is:
 1. A FLINT polypeptide comprising O-linkedoligosaccharides.
 2. A FLINT polypeptide as in claim 1 wherein saidO-linked oligosaccharides occur at Thr 174 of SEQ ID NO:1.
 3. A FLINTpolypeptide as in claim 1 wherein said O-linked oligosaccharides occurat Thr 216 of SEQ ID NO:1.
 4. A pharmaceutical formulation comprising asan active ingredient a FLINT polypeptide according to claim 1 associatedwith one or more pharmaceutically acceptable carriers, excipients, ordiluents thereof.
 5. A method for increasing the stability of a FLINTpolypeptide comprising the step of enhancing O-linked glycosylation. 6.A method to enhance resistance to proteolysis at position 218 of SEQ IDNO:1 comprising the step of increasing O-linked glycosylation atposition 216 of SEQ ID NO:1.
 7. A FLINT polypeptide compositionsubstantially comprising a polypeptide of claim 1 and a divalent metalcation.
 8. A composition as in claim 14 wherein said cation is selectedfrom the group consisting of Zn⁺², Ca⁺², Ni⁺², Mn⁺², Fe⁺², Co⁺², andCd⁺².
 9. A FLINT polypeptide comprising N-Linked oligosaccharides.